Synthesis of template-fixed β-hairpin loop mimetics

ABSTRACT

Template-fixed β-hairpin loop mimetics comprising a template corresponding to one of the structures (a), (b), (c), (d), (e), (f), (g), (h) and a template-fixed chain of 4 to 20 α-amino acid residues which, if their α-C atom is asymmetric, have L-configuration can be manufactured by a novel process which is based on a mixed solid- and solution phase synthetic strategy. If desired, this process can be modified to give the enantiomers of these template-fixed β-hairpin loop mimetics. These enantiomers are novel compounds, and many of said template-fixed β-hairpin loop mimetics themselves are also novel compounds. The template-fixed β-hairpin loop mimetics and their enantiomers can mimick flat surfaces of proteins and thus be used to probe large surface protein-protein interactions. Accordingly they can serve as lead finding tools for protein targets where it is difficult to find small-molecular-weight lead compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

-   -   This application is the National Stage filing of PCT/EP99/06369        under 35 U.S.C. §371.

The present invention relates to a reliable process for the synthesis oftemplate-fixed β-hairpin loop mimetics of the general formula

whereinZ is a chain of n α-amino acid residues which, if their α-C atom isasymmetric, have L-configuration, n being an integer from 4 to 20, thepositions of said amino acid residues in said chain being countedstarting from the N-terminal amino acid;

is one of the groups of formulae

and of salts thereof.This process is based on a mixed solid- and solution phase syntheticstrategy and comprises(a) coupling an appropriately functionalized solid support with anappropriately N-protected derivative of that amino acid which in thedesired end-product is in position a n/2, n/2+1 or n/2−1 if n is an evennumber and, respectively, in position n/2+{fraction (1/2)} or n/2−½ if nis an odd number, any functional group which may be present in saidN-protected amino acid derivative being likewise appropriatelyprotected;(b) removing the N-protecting group from the product thus obtained;(c) coupling the product thus obtained with an appropriately N-protectedderivative of that amino acid which in the desired end-product is oneposition nearer the N-terminal amino acid residue, any functional groupwhich may be present in said N-protected amino acid derivative beinglikewise appropriately protected;(d) removing the N-protecting group from the product thus obtained;(e) repeating, if necessary, steps (c) and (d) until the N-terminalamino acid residue has been introduced;(f) coupling the product thus obtained with a compound of the generalformula

wherein

is as defined above and X is an N-protecting group or, if

is to be group (a), above, alternatively

-   -   (fa) coupling the product obtained in step (d) or (e) with a        compound of the general formula III    -   wherein R¹ and X are as defined above;    -   (fb) removing the N-protecting group from the product thus        obtained; and    -   (fc) coupling the product thus obtained with an appropriately        N-protected derivative of D-proline;        (g) removing the N-protecting group from the product obtained in        step (f) or (fc);        (h) coupling the product thus obtained with an appropriately        N-protected derivative of that amino acid which in the desired        end-product is in position n, any functional group which may be        present in said N-protected amino acid derivative being likewise        appropriately protected;        (i) removing the N-protecting group from the product thus        obtained;        (j) coupling the product thus obtained with an appropriately        N-protected derivative of that amino acid which in the desired        end-product is one position farther away from position n, any        functional group which may be present in said N-protected amino        acid derivative being likewise appropriately protected;        (k) removing the N-protecting group from the product thus        obtained;        (l) repeating, if necessary, steps (j) and (k) until all amino        acid residues have been introduced;        (m) detaching the product thus obtained from the solid support;        (n) cyclising the product cleaved from the solid support;        (o) removing any protecting groups present on functional groups        of any members of the chain of amino acid residues and, if        desired, any protecting group(s) which may in addition be        present in the molecule; and        (p) if desired, converting the product thus obtained into a salt        or converting a salt thus obtained into the coon ding free        compound of formula I or into a different salt.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Solution conformation of Example-1. The D-pro-L-Pro template isat the bottom. N-atoms are in black, other atoms in gray.

DETAILED DESCRIPTION

The process of the invention can advantageously be carried out asparallel array synthesis to yield libraries of template-fixed β-hairpinloop mimetics of the above general formula I. Such parallel synthesisallows one to obtain arrays of numerous (normally 24 to 192, typically96) cyclic template-fixed peptides of general formula I in high yieldsand defined purities, minimizing the formation of dimeric and polymericby-products. The proper choice of the functionalized solid-support (i.e.solid support plus linker molecule), templates and site of cyclizationplay thereby key roles.

The β-hairpin loop mimetics of formula I can mimick flat surfaces ofproteins and thus be used to probe large surface protein—proteininteractions. They can serve as lead finding tools for protein targetswhere it is notoriously difficult to find small-molecular-weight leadcompounds. Due to the structurally and conformationally well-definedarchitecture of the β-hairpin loop mimetics of general formula I, keyamino acid residues or motifs can be integrated in conformationallylocked arrangements. By shifting these key amino acid residues or motifsalong the β-hairpin structure various conformations can be scanned(conformational scanning of key sequences). Alternatively, proteinsequences can be mapped in order to detect β-hairpin loop motifs.

This technique in summary allows to determine rapidly key amino acidsand motifs (hotspots) important for binding in large surface and flatprotein interfaces not only in their sequential but also in theirspatial arrangement. This information can ultimately be used for thedesign of small peptidomimetic drug candidates (Cunningham, B. C.;Wells, J. A. Curr. Opin. Struct. Biol. 1997, 7, 457; Obrecht, D.;Altorfer, M.; Robinson, J. A. Adv. Med. Chem. Vol. 4, 1-68, JAI PressInc., 1999).

Due to the enormous advances in genomic sciences increasing numbers ofbiologically relevant proteins (e.g. receptors, enzymes, transcriptionfactors, ligands, modulators, chaperones) are becoming available in pureform for structural and functional studies. This burst of novelbiological targets has also created a need for sources of new organicmolecules for pharmaceutical and agrochemical screening and also formore efficient screening technologies. Combinatorial and parallelchemistry have emerged in recent years to satisfy the increasing demandfor new families of novel compounds (Obrecht, D.; Villalgordo, J.-M,“Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compound Libraries”, Tetrahedron Organic ChemistSeries, Vol. 17, Pergamon, Elsevier Science, 1998).

While general screening of small-molecular-weight compounds (MG<550) hassuccessfully generated lead compounds for targets such as enzymes andreceptors with well-defined binding sites and clefts, this technologygives rather poor results when ligand binding involves large surfaceprotein-protein interactions with the corresponding receptors. Thesetargets, however, are of increasing biological and pharmaceuticalimportance and many X-ray structures of such ligands, receptors and evenligands bound to their corresponding receptors are available. Theseinclude e.g. members of the growth factor family such asplatelet-derived growth factor (PDGF) [Oefiner, C; D'Arci, A.; Winkler,F. K.; Eggimann, B.; Hosang, M. EMBO J. 1992, 11, 3921], nerve growthfactor (NGF) [Thanez, C. F.; EbendahL T.; Barbany, G.; Murray-Rust, J.;Blundell, T.; Perrson, H. Cell, 1992, 69, 320-341], epidermal growthfactor (EGF) [Biochemistry 1992, 31, 236], basic fibroblast growthfactor (b-FGF) [Biochemistry 1996, 35, 2086], transforming growth factorβII (TGF βII) [Schlunegger & Grütter, J. Mol. Biol. 1993, 231, 445],vascular endothelial growth factor (VEGF) [Müller et al., Proc. Natl.Acad. Sci. 1997, 94, 7192], and members of the cytokine family such asthe interleukines, tumor necrosis factor (TNFα and β) [Banner, D. W.;D'Arci, A.; Janes, W.; Gentz, R.; Schönfeld, H. J.; Broger, C.;Lötscher. H.; Lesslauer, W. Cell, 1993, 73, 431-445]. Moreover,chemokines [Tarby, C. M.; Saunders, J. Drug Discovery Today 1999, 4,8092; Ponath, P. D. Exp. Opin. Invest. Drugs 1998, 7, 1-16) includingmembers of the CC-family such as RANTES, MCP-1-4, Eotaxin and others,and the CXC-family such as GROα-γ, interleukine 8(Il 8) and others haveemerged as key mediators in a number of inflammatory pathologies. Inaddition, integrines [see Obrecht, D.; Altorfer, M.; Robinson, J. A.Adv. Med. Chem. Vol.4, 1-68, JAI Press Inc., 1999] play key roles incell adhesion, migration and proliferation. All these protein ligandsbind to their corresponding receptors involving one or several largesurface interactions. Moreover, X-ray crystallography and site directedmutagenesis studies highlight the importance of surface β-hairpin loopmotifs to be key in those interactions.

The anatomy of large surface protein interfaces has recently beenanalysed and the average contact surface was determined to be typically600-900 A². The free energy of binding is not evenly distributed acrossthe interfaces; instead, there are hot spots of binding energy made upof a small subset of residues in the dimer interface. These hot spotsare enriched in tryptophan (Trp), tyrosine (Tyr) and arginine (Arg), andare surrounded by energetically less important residues that are mostlikely serving to occlude solvent from the hot spot [Bogan, A. A.;Thorn, K. S. J. Mol. Biol. 1998, 280, 1-9]. Occlusion of solvent isbelieved to be a necessary condition for highly energetic interactions.The β-hairpin loop motif offering two opposite β-sheet surfaces (e.g. ahydrophobic and a hydrophilic face) for possible binding interactions isideally suited to meet these criteria for surface interactions.

The β-hairpin motif is very abundant in nature and occurs on the surfaceof many protein ligands and in the hypervariable domains of antibodies.The β-hairpin motif consists of two antiparallel β-strands linked by ashort loop or turn and have been classified depending on the H-bondingnetwork [Sibanda, B. L.; Blundell, T. L.; Thornton, J. M. J. Mol. Biol.1989, 206, 759-777]. One example, par excellence, is found in theantigen binding sites of antibodies [Padlan, E. A. Mol. Immunol. 1994,31, 169-217], which are composed of amino acid residues located in sixso-called hypervariable loops or complementarity-determining-regions(CDR's), three each from the heavy- and light-chain variable regions(v_(H) and v_(L)). Of the six CDR loops in antibodies of the Ig family,four may be classified as β-hairpins connecting adjacent antiparallelβ-sheets, two from the V_(L) domain, L₂ and L₃, and two from the V_(H)domain, H₂ and H₃. Recent estimates suggest that a large majority of L₁,L₂, L₃, H₁ and H₂ hypervariable regions maybe classified into one of 18different canonical conformations [Chothia, C.; Lesk, A.; Gherardi, E.;Tomlinson, J. M.; Walter, G.; Marks, J. G.; Llewelyn, M. B.; Winter, G.J. Mol. Biol. 1992, 227, 799-817; Martin, A. C.; Thornton, J. M. J. Mol.Biol. 1996, 263, 800-815; Al-Lazikani, B.; Lesk, A.; Chothia, C. J. Mol.Biol. 1997, 273, 927-948].

The present invention provides a reliable process for the synthesis oftemplate-fixed cyclic peptides of general formula I which mimick thevarious naturally occurring β-hairpin conformations, especially thosepresent in growth factors, cytokines and chemokines, integrines andantibodies (see e.g. Figure, Example 1). Template structurescorresponding to above formulae (a) through (h) have been shown tostabilize the H-bond network present in β-hairpins [e.g. for (a): Spaethet al. Helv. Chim. Acta 1998, 81, 1726; Favre, M.; Moehle, K; Jiang, L.;Pfeiffer, B.; Robinson, J. A. J. Am. Chem. Soc. 1999, 121, 2679-2685;for (b): Emery et al., J Chem. Soc. Chem. Comm. 1996, 2155; Bisang etal. J. Am. Chem. Soc. 1998, 120, 7439; for (c): Pfeifer, M. J. Chem.Soc. Chem. Commun. 1998, 1977; for (d): Pfeifer et al. Helv. Chim. Acta1997, 80, 1513; for (e): Beeli et al. Helv. Chim. Acta 1996, 79, 2235;and for (f) and analogues: Müller, K.; Obrecht, D.; Knierzinger, A.;Stankovic, C; Spiegler, C.; Trzeciak, A.; Englert, G.; Labhardt, A. M.;Schönholzer, P. Perspectives in Medicinal Chemistry; Testa, B., Kyburz,E., Fuhrer, W., Gyger, R., Eds.; Verlag Helv. Chim. Acta: Basel, 1993;pp 513-531); for (g) and (h) and analogues: Müller, K; Obrecht, D.;Knierzinger, A.; Spiegler, C.; Bannwarth, W.; Trzeciak, A.; Englert, G.;Labhardt, A.; Schönholzer, P. Perspectives in Medicinal Chemistry,Editor Testa, B.; Kyburz, E.; Fuhrer, W.; Giger, R., Weinheim, New York,Basel, Cambridge: Verlag Helvetica Chimica Acta, 1993, 513-531;Bannwarth, W.; Gerber, F.; Grieder, A.; Knierzinger, A.; Müller, K.;Obrecht. D.; Trzeciak, A. Can Pat. Appl. CA2101599].

As stated above, the process of the invention takes advantage of a mixedsolid- and solution phase synthetic approach which can be performed in aparallel array of e.g. 24-192, preferably 96, reactions, and providesthe template-fixed cyclic peptides of general formula I in good yieldsand defied purities, ready for screening, thereby minimizing the amountof dimeric and polymeric impurities, which tend to give false positivehits in the screening process. This process is clearly superior topreviously described syntheses of cyclic peptides by Bannwarth, W.;Gerber, F.; Grieder, A.; Knierzinger, A.; Müller, K.; Obrecht. D.;Trzeciak, A. Can. Pat. Appl. CA2101599. The proper choice of resin andloading capacity, linker molecule, template and site of cyclization arekey for obtaining high yields and reliable purities of β-hairpin loopmimetics. The templates thereby do not only stabilise the conformationsof the final products, but they significantly enhance the rate ofcyclization to the monomer, most probably by β-hairpin type H-bondinduction.

Due to the well-defined architecture of the various β-hairpin loopmimetics of general formula I key amino acid residues and motifs can belocked in various conformations by shifting the sequence along theβ-hairpin backbone (“conformational scanning of biologically activesequences”). Alternatively, protein sequences can be mapped by usingthis approach in order to detect β-hairpin conformations. Thus, thisβ-hairpin mimetics approach provides a technique to detect hot spots ofhigh energy interactions in protein interfaces in three-dimensionalarrangement. This information should ultimately be transferable into thedesign of small peptidomimetic molecules.

As used in the present description, the term “lower alkyl”, taken aloneor in combinations such as “aryl-lower alkyl”, embraces straight chainor branched saturated hydrocarbon residues with up to 7, preferably upto 4 carbon atoms such as methyl ethyl n-propyl, isopropyl, n-butylisobutyl, sec.-butyl, tert.-butyl and the like. The term “lower alkoxy”embraces alkyloxy groups in the sense of the above description of thetern “lower alkyl”, such as methoxy, ethoxy, n-butoxy, and the like. Theterm “aryl” embraces the phenyl residue and substituted phenyl residues,especially mono- or disubstituted phenyl residues, with lower alkyl orlower alkoxy groups or halogen atoms primarily coming into considerationas substituents. The term “halogen” denotes the four forms fluorine,chlorine, bromine and iodine unless indicated otherwise. The term “acyl”embraces residues of aliphatic and aromatic carboxylic acids, primarilyon the one hand lower alkanoyl groups such as acetyl, propionyl, butyryland the like, which can be substituted, for example by carboxy or loweralkoxycarbonyl, as is the case e.g. in 4-carboxybutyryl,4-methoxycarbonylbutyryl or the like, and on the other hand aroyl groupssuch as the benzoyl group and substituted benzoyl groups, especiallymono or disubstituted benzoyl groups, with lower alkyl or alkoxy groupsor halogen atoms primarily coming into consideration as substituents.The term “substituted lower alkyl” embraces lower allyl groups which aresubstituted by protected amino, lower alkoxy, COOR¹⁰ (in which R¹⁰ is asabove), carboxamido or N-lower alkylcarboxamido such asphthalimidomethyl, methoxymethyl, methoxyethyl and the like. The term“protected amino” embraces on the one hand residues such as phthalimido(“Pt”) and the like and on the other hand residues of the formula—NH—R¹¹ in which R¹¹ can signify any appropriate N-protecting group suchas benzyloxycarbonyl (“Z”), tert.-butyloxycarbonyl (“Boc”),9-fluorenylmethoxycarbonyl (“Fmoc”), allyloxycarbonyl (“Alloc”),trimethylsilylethoxycarbonyl (“Teoc”), trichloroethoxycarbonyl (“Tcc”),o-nitrophenylsulfonyl (“Nps”) and the like.

As amino acid residues there primarily come into consideration thosewhich are derived from natural α-amino acids. Hereinafter there is givena list of such amino acids which, or the residues of which, are suitablefor the purposes of the present invention, the abbreviationscorresponding to generally adopted usual practice.

Ala A L-Alanine Arg R L-Arginine Asn N L-Asparagine Asp D L-Asparticacid Cys C L-Cysteine Glu E L-Glutamic acid Gln Q L-Glutamine Gly GGlycine His H L-Histidine Ile I L-Isoleucine Leu L L-Leucine Lys KL-Lysine Met M L-Methionine Phe F L-Phenylalanine Pro P L-Proline Ser SL-Serine Thr T L-Threonine Trp W L-Tryptophan Tyr Y L-Tyrosine Val VL-Valine

Other α-amino acids which, or the residues of which, are suitable forthe purposes of the present invention include

C₄al L-3-Cyclobutylalanine C₅al L-3-Cyclopentylalanine C₆alL-3-Cyclohexylalanine aIle L-Alloisoleucine Nal L-3-(1-Naphthylalanine)Nle L-Norleucine Nva L-Norvaline Orn L-Ornthine Orn(CHO)N⁵-Formyl-L-ornithine L-Phg L-Phenylglycine Tza L-3-(2-Thazolyl)alanine

It will be appreciated that the compound of the above general formulaIII, i.e. one of the two building blocks of the template structurecorresponding to the above formula (a), is a derivative of L-proline(L-Pro, ^(L)P), whilst the second of these building blocks is a residueof D-proline (D-Pro, ^(D)P).

Preferred values for n, i.e. the number of amino acid residues presentin the chain Z, are, in general, 4-16. Particularly preferred values ofn are 6, 10 and 14 in case the template structure corresponds to theabove formula (b) or (c) or (d), and 4, S, 6, 8, 12 and 16 in the caseof the other template structures, i.e. those corresponding to the aboveformulae (a), (e), (f), (g) and (h).

Advantageously the chain Z consist of, or contains, a key sequence oftwo, thee four, five, six or occasionally up to ten amino acid residues,the two terminal members of which are “constant” (“k”) whilst any othermembers are either “constant”, too or “variable”(“x”), in all possiblecombinations or permutations. The two terminal “constant” members can bethe same or different, and the same applies to any remaining “constant”and/or to any “variable” members.

Particularly suitable “constant” members (“k”) are Trp, Arg, Tyr, Ile,Asp, His, Lys, Glu and Thr, further suitable “constant” members (“k”)are Gln, Phe, Met and Ser, and suitable “variable” members (“x”) areAla, Orn, Leu and Val.

Key sequences of two, three, four, five and six amino acid residues, canbe schematically depicted as follows:

dipeptide -k¹-k²- tripeptide -k¹-k²-k³- -k¹-x¹-k²- tetrapeptide-k¹-k²-k³-k⁴- -k¹-x¹-k²-k³- -k¹-k²-x¹-k³- -k¹-x¹-x²-k²- pentapeptide-k¹-k²-k³-k⁴-k⁵- -k¹-x¹-k²-k³-k⁴- -k¹-k²-x¹-k³-k⁴- -k¹-k²-k³-x¹-k⁴--k¹-x¹-x²-k²-k³- -k¹-k²-x¹-x²-k³- -k¹-x¹-k²-x²-k³- -k¹-x¹-x²-x³-k²-hexapeptide -k¹-k²-k³-k⁴-k⁵-k⁶- -k¹-x¹-k²-k³-k⁴-k⁵- -k¹-k²-x¹-k³-k⁴-k⁵--k¹-k²-k³-x¹-k⁴-k⁵- -k¹-k²-k³-k⁴-x¹-k⁵- -k¹-x¹-x²-k²-k³-k⁴--k¹-k²-x¹-x²-k³-k⁴- -k¹-k²-k³-x¹-x²-k⁴- -k¹-x¹-k²-x²-k³-k⁴--k¹-k²-x¹-k³-x²-k⁴- -k¹-x¹-x²-x³-k²-k³- -k¹-k²-x¹-x²-x³-k³--k¹-x¹-k²-x²-x³-k³- -k¹-x¹-x²-k²-x³-k³- -k¹-x¹-x²-x³-x⁴-k²-

Certain key sequences arm known to occur in important physiologicallyactive peptides, such as

R G D in fibronectin (FN), vitronectin (VN), osteopontin, collagens,thrombospondin, fibrinogen (Fg), von Willebrand factor (vWF), seeObrecht, D.; Altorfer, M.; Robinson, J. A. Adv. Med. Chem. Vol 4, 1-68,JAI Press Inc., 1999 E L R in C X C chemokines, see Saunders, J.; Tarby,C. M. Drug Discovery Today, 1999, 4, 80-92 R K K see J. Biol. Chem.1999, 274, 3513 K G F see Prot. Sci. 1998, 7, 1681-1690 V R K K [SEQ IDNO:1] in Platelet-Derived Growth Factor (PDGF), see Ross, R.; Raines, E.W.; Bowden-Pope, D. F. Cell, 1986, 46, 155-159 K K Y L [SEQ ID NO:2] inVIP (vasointestinal peptide) showing neuroprotective properties againstβ-amyloid neurotoxicity, see Proc. Natl. Am. Soc. USA 1999, 96,4143-4148 W L D V [SEQ ID NO:3] in integrin α₄β₁ see Europ. J. Biol.1996, 242, 352-362 and Int. J. Pept. Prot. Res. 1996, 47, 427-436 Y I RL P [SEQ ID NO:4] in Factor Xa inhibitors, see Al Obeidis, F.; Ostrem,J. A. Drug Discovery Today 1998, 3, 223-231 Y J G S R [SEQ ID NO:5] inlaminine, see EMBO. J. 1984, 3, 1463 I K V A V [SEQ ID NO:6] see Cell1987, 88, 989 P P R X X W [SEQ ID NO:7] see J. Biol. Chem. 1998, 273,11001-11006 & 11007-11011 I Y Y K D G A L K Y [SEQ ID NO:8] see BiochemSoc. Trans. 1997, 29, 387-392

If desired, the process of the invention can be modified to give theenantiomers of the compounds of the general formula I. To this effectall amino acids which have an asymmetric α-carbon atom are used in theirD-Form and the enantiomer of a template corresponding to structure (a),(b), (c), (d) or (e) or a template corresponding to formula (f), (g or(h) is used in step (f) and, respectively, the enantiomer of a compoundof formula III is used in step (fa) and a derivative of L-proline isused in step (fc).

Suitable protecting groups for amino acids and, respectively, for theirresidues are, for example,

-   -   for the amino group (as is present e.g. also in the side-chain        of lysine)

Z benzloxycarbonyl Boc tert-butyloxycarbonyl Fmoc9-fluorenylmethoxycarbonyl Alloc allyloxycarbonyl Teoctrimethylsilylethoxycarbonyl Tcc trichloroethoxycarbonyl Npso-nitrophenylsulfonyl; Tr triphenymethyl or trityl

-   -   for the carboxyl group (as is present e.g. also in the        side-chain of aspartic and glutamic acid) by conversion into        esters with the alcohol components

tBu tert.-butyl Bn benzyl Me methyl Ph phenyl Pac Phenacyl Allyltrimethylsilylethyl trichloroethyl;

-   -   for the guanidino group as is present e.g. in the side-chain of        arginine

Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl Ts tosyl (i. e.p-toluenesulfonyl) Z benzyloxycarbonyl Pbfpentamethyldihydrobenzofuran-5-sulfonyl

-   -   for the hydroxy group (as is present e.g. in the side-chain of        threonine and serine)

tBu tert.-butyl Bn benzyl Tr trityl

-   -   and for the mercapto group (as is present e.g. in the side-chain        of cysteine)

tBu tert.-butyl Bn benzyl Tr trityl Mtr 2-methoxytrityl.

The functionalized solid support is conveniently derived frompolystyrene crosslinked with, preferably 1-5%, divinybenzene to;polystyrene coat ed with polyethyleneglycol spacers (Tentagel^(R)); andpolyacrylamide resins (see also Obrecht, D.; Villalgordo, J.-M,“Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compound Libraries”, Tefrahedron OrganicChemistry Series, Vol. 17, Pergamon, Elsevier Science, 1998).

The solid support is functionalized by means of a linker, i.e. abifunctional spacer molecule which contains on one end an anchoringgroup for attachment to the solid support and on the other end aselectively cleavable functional group used for the subsequent chemicaltransformations and cleavage procedures. For the purposes of the presentinvention the linker must be designed to eventually release the carboxylgroup under mild acidic conditions which do not affect protecting groupspresent on any functional group in the side-chains of the various aminoacids. Linkers which are suitable for the purposes of the presentinvention form acid-labile esters with the carboxyl group of the aminoacids, usually acid-labile benzyl, benzhydryl and trityl esters;examples of linker structures of this kind include3-methoxy-4-hydroxymethylphenoxy (Sasrin linker),4-(2,4-dimethoxyphenyl-hydroxymethyl)-phenoxy (Rink linker),4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB linker), trityland 2-chlorotrityl.

When carried out as a parallel array synthesis the process of theinvention can be advantageously carried out as described hereinbelow butit will be immediately apparent to those skilled in the art how thisprocedure will have to be modified in case it is desired to synthesizeone single compound of the above formula I.

A number of reaction vessels (normally 24 to 192, typically 96) equal tothe total number of compounds to be synthesized by the parallel methodare loaded with 25 to 1000 mg, preferably 100 mg, of the appropriatefunctionalized solid support, preferably 1 to 3% cross linkedpolystyrene or tentagel resin.

The solvent to be used must be capable of swelling the resin andincludes, but is not limited to, dichloromethane (DCM),dimethylformamide (DMF), N-methylpyrrolidinone (NMP), dioxane, toluene,tetrahydrofuran (THF), ethanol (EtOH), trifluoroethanol (TFE),isopropylalcohol and the like. Solvent mixtures containing as at leastone component a polar solvent (e.g. 20% TFE/DCM, 35% THF/NMP) arebeneficial for ensuring high reactivity and salvation of the resin-boundpeptide chains (Fields, G. B., Fields, C. G., J. Am. Chem. Soc. 1991,113, 4202-4207).

With the development of various linkers that release the C-terminalcarboxylic acid group under mild acidic conditions, not affectingacid-labile groups protecting functional groups in the side chain(s),considerable progresses have been made in the synthesis of protectedpeptide fragments. The 2-methoxy-4-hydroxybenzylalcohol-derived linker(Sasrin^(R) linker, Metier et al., Tetrahedron Lett. 1988, 29 4005-4008)is cleavable with diluted trifluoroacetic acid (0.5-1% TFA in DCM) andis stable to Fmoc deprotection conditions during the peptide synthesis,Boc/tBu-based additional protecting groups being compatible with thisprotection scheme. Other linkers which are suitable for the process ofthe invention include the super acid labile4-(2,4-dimethoxyphenyl-hydroxymethyl)-phenoxy linker (Rink linker, Rink,H. Tetrahedron Lett. 1987, 28, 3787-3790), where the removal of thepeptide requires 10% acetic acid in DCM or 0.2% trifluoroacetic acid inDCM; the 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid-derived linker(HMPB-linker, Flörshcimer & Riniker, Peptides 1991, 1990 131) which isalso cleaved with 1% TFA/DCM in order to yield a peptide fragmentcontaining all acid labile side-chain protective groups; and, inaddition, the 2-chlorotritylchloride linker (Barlos et al., TetrahedronLett. 1989, 30, 3943-3946), which allows the peptide detachment using amixture of glacial acetic acid/trifluoroethanol/DCM (1:2:7) for 30 min.

The 9-fluorenylmethoxycarbonyl-(Fmoc)-protected amino acid derivativesare preferably used as the building blocks for the construction of thetemplate-fixed β-hairpin loop mimetics of formula I. For thedeprotection, i.e. cleaving off of the Fmoc group, 20% piperidine in DMFor 2% DBU/2% piperidine in DMF can be used

The quantity of the reactant, i.e. of the amino acid derivative, isusually 1 to 20 equivalents based on the milliequivalents per gram(meq/g) loading of the functionalized solid support (typically 0.1 to2.85 meq/g for polystyrene resins) originally weighed into the reactiontube. Additional equivalents of reactants can be used if required todrive the reaction to completion in a reasonable time. The reactiontubes, in combination with the holder block and the manifold, arereinserted into the reservoir block and the apparatus is fastenedtogether. Gas flow through the manifold is initiated to provide acontrolled environment, for example, nitrogen, argon, air and the like.The gas flow may also be heated or chilled prior to flow through themanifold. Heating or cooling of the reaction wells is achieved byheating the reaction block or cooling externally with isopropanol/dryice and the like to bring about the desired synthetic reactions.Agitation is achieved by shaking or magnetic stirring (within thereaction tube). The preferred workstations (without, however, beinglimited thereto) are Labsource's Combi-chem station and MultiSynTech's-Syro synthesizer.

Amide bond formation requires the activation of the carboxyl group forthe acylation step. When this activation is being carried out by meansof the commonly used carboddiimides such as dicyclohexylcarbodiimide(DCC, Sheehan & Hess, J. Am. Chem. Soc. 1955, 77, 1067-1068) ordiisopropylcarbodiimide (DIC, Sarantakis et al Biochem. Biophys. Res.Commun.1976, 73, 336-342), the resulting dicyclohexylurea is insolubleand, respectively, diisopropylurea is soluble in the solvents generallyused. In a variation of the carbodiimide method 1-hydroxybenzotriazole(HOBt, König & Geiger, Chem. Ber 1970, 103, 788-798) is included as anadditive to the coupling mixture. HOBt prevents dehydration, suppressesracemization of the activated amino acids and acts as a catalyst toimprove the sluggish coupling reactions. Certain phosphonium reagentshave been used as direct coupling reagents, such asbenzotriazole-1-yl-oxy-tris-(dimethylamino) phosphoniumhexafluorophosphate (BOP) (Castro et al., Tetrahedron Lett. 1975, 14,1219-1222; Synthesis, 1976, 751-752), orbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexaflurophosphate(Py-BOP, Coste et al., Tetrahedron Lett. 1990, 31, 205-208), or2-(1H-benzotriazole-1-yl-)1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU), or hexaflurophosphate (HBTU, Knorr et al., Tetrahedron Lett.1989, 30, 1927-1930); these phosphonium reagents are also suitable forin situ formation of HOBt esters with the protected amino acidderivatives. More recently diphenoxyphosphoryl azide (DPPA) orO-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TATU) orO-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU)/7-aza-1-hydroxy benzotriazole (HOAt, Carpinoet al., Tetrahedron Lett. 1994, 35, 2279-2281) have also been used ascoupling reagents.

Due to the fact that near-quantitative coupling reactions are essentialit is desirable to have experimental evidence for completion of thereactions. The ninhydrin test (Kaiser et al., Anal. Biochemistry 1970,34, 595), where a positive calorimetric response to an aliquot ofresin-bound peptide indicates qualitatively the presence of the primaryamine, can be easily and quickly performed after each coupling step.Fmoc chemistry allows the spectrophotometric detection of the Fmocchromophore when it is released with the base (Meienhofer et al., Int.J. Peptide Protein Res. 1979, 13, 35-42).

The resin-bound intermediate within each reaction tube is washed cleanof excess of retained reagents, of solvents, and of by-products byrepetitive exposure to clean solvent(s) by one of the two followingmethods:

-   -   1) The reaction wells are filled with solvent (preferably 5 ml),        the reaction tubes, in combination with the holder block and        manifold, are immersed and agitated for 5 to 300 minutes,        preferably 15 minutes, and drained by gravity followed by gas        pressure applied through the manifold inlet (while closing the        outlet) to expel the solvent;    -   2) The manifold is removed from the holder block, aliquots of        solvent (preferably 5 ml) are dispensed through the top of the        reaction tubes and drained by gravity through a filter into a        receiving vessel such as a test tube or vial.

Both of the above washing procedures are repeated up to about 50 times(preferably about 10 times), monitoring the efficiency of reagent,solvent, and byproduct removal by methods such as TLC, GC, orvisualization of the wash filtrates.

The above described procedure of reacting the resin-bound compound withreagents within the reaction wells followed by removal of excessreagents, by-products, and solvents is repeated with each successivetransformation until the final resin-bound compound is prepared.

Detachment of the fully protected linear peptide from the solid supportis achieved by immersion of the reaction tubes, in combination with theholder block and manifold, in reaction wells containing a solution ofthe cleavage reagent (preferably 3 to 5 ml). Gas flow, temperaturecontrol agitation, and reaction monitoring are implemented as describedabove and as desired to effect the detachment reaction. The reactiontubes, in combination with the holder block and manifold, aredisassembled from the reservoir block and raised above the solutionlevel but below the upper lip of the reaction wells, and gas pressure isapplied through the manifold inlet (while closing the outlet) toefficiently expel the final product solution into the reservoir wells.The resin remaining in the reaction tubes is then washed 2 to 5 times asabove with 3 to 5 ml of an appropriate solvent to extract (wash out) asmuch of the detached product as possible. The product solutions thusobtained are combined, taking care to avoid cross-mixing. The individualsolutions/extracts are then manipulated as needed to isolate the finalcompounds. Typical manipulations include, but are not limited to,evaporation, concentration, liquid/liquid extraction, acidification,basification, neutralization or additional reactions in solution.

The solutions containing fully protected linear peptide derivativeswhich have been cleaved off from the solid support and neutralized witha base, are evaporated, then cyclization is effected in solution usingsolvents such as DCM, DMF, Dioxane, THF and the like. Various couplingreagents which were mentioned earlier can be used for the cyclization.The duration of the cyclization is about 6-48 hours, preferably about 24hours. The progress of the reaction is followed, e.g. by RP-HPLC(Reverse Phase High Performance Liquid Chromatography). Then the solventis removed by evaporation, the fully protected cyclic peptide derivativeis dissolved in a solvent which is not miscible with water, such as DCM,and the solution is extracted with water or a mixture of water-misciblesolvents, in order to remove any excess of the coupling reagent.

The fully protected cyclized peptide derivative is treated with 95% TFA,2.5% H₂O, 25% TIS or another combination of scavengers for effecting thecleavage of protecting groups. The cleavage reaction time is commonly 30minutes to 12 hours, preferably 2 hours. Thereafter most of the TFA isevaporated and the product is precipitated with ether/hexane (1:1) orother solvents which are suitable therefor. After careful removal of thesolvent, the cyclic peptide derivative obtained as end-product can beisolated. Depending on its purity, this peptide derivative can be useddirectly for biological assays, or it has to be further purified, forexample by preparative HPLC.

The end-products, i.e. the compounds of formula I, can be individuallytested for biological activity once they have been isolated andcharacterized For example, the following Solid-Phase assay can becarried out.

Direct immobilization of platelet-derived growth factor β(PDGFR-β) isperformed by overnight incubation in immunosorbent 96-well plates (Nunc)at 4° C. using 100 ng of purified protein in 100 μl of 15 mM Na₂CO₃, 35mM NaHCO3, pH 9.6. The plates are washed once with tris-buffered saline(TBS, 20 mM Tris-HCl, 150 mM NaCl, pH 7.4), and nonspecific adsorptionis blocked by at least 1 h of incubation with TBS plus 1% bovine serumalbumin (BSA). Following washing with TBS plus 0.1% Tween, 3000 cpm of¹²⁵I-PDGF-BB and increasing amounts of unlabeled PDGF-BB or the peptidederivative of formula I are added to duplicate wells and incubated for 3h at room temperature in 0.1% Tween, 1 mM CaCl₂, 1 mM MgCl₂ and 1% BSA.The plates are washed three times with TBS plus 0.1% Tween, and boundligand is removed with 0.1 M citric acid, pH 2.5, prior to counting in aγ-counter.

Some of the compounds embraced by general formula I have already beendescribed but the remaining of these compounds are novel and form partof the present invention, namely those of formula I with the provisosthat if

is(i) group (a) and R¹ is hydrogen, then Z is other than

-   -   -Val-Lys-Asn-Tyr-Gly-Val-Lys-Asn-Ser-Glu-Trp-Ile-[SEQ ID NO:9],    -   -Val-Lys-Asn-Tyr-Gly-Val-Lys-Asn-Ser-Glu-Trp-Thr-[SEQ ID NO:10],    -   -Gly-Arg-Gly-Asp-(SEQ ID NO:1)],    -   -Arg-Gly-Asp-Gly-[SEQ ID NO:12],    -   -Phe-Tyr-Thr-Gly-Thr-[SEQ ID NO:13],    -   -Tyr-Arg-Asp-Ala-Met-[SEQ ID NO:14],    -   -Asn-Thr-Tyr-Ser-Gly-Val-[SEQ ID NO:15],    -   -Trp-Asp-Asp-Gly-Ser-Asp-[SEQ ID NO:16] and    -   -Leu-Trp-Tyr-Ser-Asn-His-Trp-Val-[SEQ ID NO:17];        (ii) group (b) and R² is hydrogen or CH₂-COOH, or group (c) and        R³ is benzoyl, or group (d), or group (e), then Z is other than        -Ala-Asn-Pro-Asn-Ala-Ala-[SEQ ID NO:18];        (iii) group (b) and R² is hydrogen, then Z is other than        -Ala-Arg-Gly-Asp-[SEQ ID NO:19];        (iv) group (f), R⁴ is methyl, R⁵ is methoxy and R⁶ and R⁷ each        are hydrogen, then Z is other than    -   -Val-Ala-Ala-Phe-Leu-Ala-Leu-Ala-[SEQ ID NO:20],    -   -Arg-Gly-Asp-Val-[SEQ ID NO:21],    -   -Ala-Thr-Val-Gly-[SEQ ID NO:22],    -   -Glu-Arg-Gly-Asp-Val-Tyr-[SEQ ID NO:23],    -   -Ile-Ala-Arg-Gly-Asp-Phe-Pro-Asp [SEQ ID NO:24],    -   -Ala-Arg-Ile-Ala-Arg-Gly-Asp-Phe-Pro-Asp-Asp-Arg-[SEQ ID NO:25],    -   -Ala-Arg-Gly-Asp-Phe-Pro-[SEQ ID NO:26]    -   -Arg-Gly-Asp-Phe-[SEQ ID NO:27] and    -   -Arg-Ile-Ala-Arg-Gly-Asp-Phe-Pro-Asp-Asp-[SEQ ID NO:28];        (v) group (g), R⁸ is methyl and R⁹ is methyl or n-hexyl, or        group (h), R⁸ is ethyl and R⁹ is ethyl, then Z is other than        -Arg-Gly-Asp-Val-[SEQ ID NO:21];        (vi) group (9), R⁸ is methyl and R⁹ is methyl or benzyl, then Z        is other than -Gly-Gly-Ala- Gly-[SEQ ID NO:29];        (vii) group (g), R⁸ is methyl and R⁹ is methyl, then Z is other        than -Gly-Asp-Gly-Gly-[SEQ ID NO:30]; and        (viii) group (g), R⁸ is methyl and R⁹ is n-hexyl, then Z is        other than -Val-Arg-Lys-Lys [SEQ ID NO:1].

The enantiomers of all compounds of formula I are novel and also formpart of the present invention.

The compounds of formula II incorporating structures (a) to (h) and thecompounds of formula III can be prepared as shown in the followingReaction Schemes. Throughout these Reaction Schemes the N-protectinggroup X present in the compounds of formulae II and III is indicated tobe Fmoc, the preferred value for X, but it will be appreciated thatcorresponding compounds carrying as X other N-protecting groups can beprepared in a similar way.

IV→Vi: Treatment of IV with a dehydrating reagent such as thionylchloride inmethanol at an elevated temperature, conveniently at reflux.ii: Introduction of Boc, e.g. using di-tert-butyl dicarbonate andtriethylamine in a suitable solvent such as dichloromethane; any othersuitable N-protecting group (not shown in Reaction Scheme 1) can beintroduced in an analogous manner.iii: Reaction of formed product with phthalimide, diethyldiazodicarboxylate and triphenylphoshine under standard Mitsunobuconditions (Mitsunobu, O.; Wada, M.; Sano, T. J. J. Am. C Soc. 1972, 94,672) to conveniently yield V.V→VIiv: Cleavage of the phthalimide group, suitably by treatment of V withhydrazine hydrate in a suitable solvent, such as ethanol at an elevatedtemperature, conveniently at about 80° C.v: Standard protection of the 3-amino group.vi: Saponification of the methyl ester group using e.g. a suitable basicreagent such as lithium hydroxide in methanol and water.vii: The tert-butoxycarbonyl group is subsequently cleaved off usingreagents such as trifluoroacetic acid in dichloromethane or 4Nhydrochloric acid in dioxane.viii: The formed amino acid is conveniently protected with reagents suchas 9-fluorenylmethoxcarbonyl chloride or 9-fluorenylmethoxcarbonylsuccinimide using a base such as sodium carbonate or triethylamine in asuitable solvent or mixture of solvents such as dioxane and water, ordichloromethane to yield VI.

V→VII

i: Treatment of V with trifluoracetic acid in dichloromethane.

VII→VIII

ii: VII is coupled under standard peptide coupling conditions withZ-Asp(tBu)OH in DMF with reagents such as HBTU and 1-hydroxybenztriazole(HOBt) with a base such as diisopropylethylamine to yield VIII.

VIII→IX

iii: Removal of the Z-group, conveniently by hydrogenation using H₂ anda catalyst such as Palladium on charcoal, in solvents such as ethanol,DMF and ethyl acetate.

iv: The phthalimide group is cleaved off from the resulting product,conveniently by treatment with hydrazine in a suitable solvent such asethanol at an elevated temperature, suitably at about 80° C.

v: The formed amino acid is conveniently protected with reagents such as9-fluorenylmethoxcarbonyl chloride or 9-fluorenylmethoxcarbonylsuccinimide using a base such as sodium carbonate or triethylamine in asuitable solvent or mixture of solvents such as dioxane and water, ordichloromethane to yield IX as described by Bisang, C.; Weber, C.;Robinson, J. A. Helv. Chim. Acta 1996, 79, 1825-1842.

IV→X

i: Treatment of IV with a dehydrating reagent such as thionyl chloridein a suitable solvent such as methanol at an elevated temperatureconveniently at reflux.

ii: The resulting amino acid ester is N-protected under standardconditions for introducing the Z group, e.g. using benzyloxycarbonylchloride and triethylamine in a suitable solvent such asdichloromethane.

iii: The Z-protected amino acid methyl ester is treated withtrimethylsilylchloride and a base such as triethylamine in a solventsuch as tetrahydrofuran, cooled, conveniently to about −78° C., followedby reaction with a strong base such as lithium diisopropylamide orlithium hexamethyldisilylazide and tert.-butyl bromoacetate yielding Xas a mixture of diastereomers as described by Bisang, C.; Jiang, L.;Freund, E.; Emery, F.; Bauch, C.; Matile, H,; Pluschke, G.; Robinson, J.A. J. Am. Chem. Soc. 1998, 120, 7439-7449; Emery, F.; Bisang, C.; Favre,M.; Jiang, L.; Robinson, J. A J. Chem Soc. Chem. Commun. 1996,2155-2156.

X→XI

iv: Reaction of X with phthalimide, diethyl diazadicarboxylate andtriphenylphoshine under standard Mitsunobu conditions (Mitsunobu, O.;Wada, M.; Sano, T. J. J. Am. Chem. Soc. 1972, 94, 672.

v: The resulting product is hydrogenated using H₂ and a suitablecatalyst such as Palladium on charcoal in a solvent such as ethylacetate, DMF or ethanol; subsequently separation of diastereomers takesplace.

XI→XII

vi: XII is coupled with Fmoc-Asp(allyl)OH under standard peptidecoupling conditions using reagents such as HATU, HOAt and a base such asdiisopropylethylamine in a suitable solvent such as DMF.

vii: Cyclization, conveniently with DBU in DMF.

XII→XIII

viii: The phthalimide group is cleaved off from resulting product,conveniently by hydrazinolysis, e.g. treatment with methylhydrazine in asuitable solvent such as DMF.

ix: The formed product is conveniently protected with reagents such as9-fluorenylmethoxcarbonyl chloride or 9-fluorenylmethoxcarbonylsuccinimide using a base such as sodium carbonate or triethylamine in asuitable solvent or mixture of solvents such as dioxane and water, ordichloromethane.

XIII→XIV

x: Standard removal of an allyl ester group using e.g. Palladium(0) ascatalyst

XV→XVI

i: XV (obtainable from Vitamin C as described by Hubschwerlen, C.(Synthesis 1986, 962) is treated with phthalimide, diethyldiazodicarboxylate and triphenylphoshine under standard Mitsunobuconditions (Mitsunobu, O.; Wada, M.; Sano, T. J. J. Am. Chem. Soc. 1972,94, 672).

ii: The phthalimide group is cleaved off from the product, convenientlyby hydrazinolysis, e.g. by treatment with methylhydrazine in a suitablesolvent such as DMF.

iii: The amino group is protected by treatment with a benzoylatingreagent such as benzoic acid anhydride or benzoylchloride and a basesuch as triethylamine or 4-dimethylaminopyridine in a suitable solventsuch as dichloromethane or DMF.

iv: Removal of the 2,4-dimethoxybenzyl group, e.g. with K₂S₂O₈ andNa₂HPO₄ in aqueous acetonitrile at an elevated temperature, e.g. atabout 80° C.

v: Introduction of a tert.-butoxycarbonyl group using e.g.di-tert.-butyloxycarbonyl dicarbonate, triethylamine and a catalyticamount of 4-dimethylaminopyridin in a suitable solvent such asdichloromethane.

vi: Reaction with aqueous sodium carbonate in tetrahydrofuran followedby acidification.

vii: Esterification of the carboxylic acid group, conveniently withdiazomethane in a suitable solvent such as diethylether.

viii: Removal of the Z-group, conveniently by hydrogenation with H₂ inthe presence of a catalyst such as Palladium on charcoal in a solventsuch as DMF to yield XVI as described by Pfeifer, M.; Robinson, J. A. J.Chem. Soc. Chem. Commun. 1998, 1977.

XVI→XVII ix: XVI is coupled under standard peptide coupling conditionswith Z-Asp(tBu)OH in DMF with reagents such as HBTU and1-hydroxybenztriazole with a base such as diisopropylethylamine to yieldXVII as described by Pfeifer, M.; Robinson, J. A. J. Chem. Soc. Chem.Commun. 1998, 1977.

XVII→XVIII

x: Removal of the Z-group, e.g. by hydrogenation using H₂ and a catalystsuch as Palladium on charcoal under standard conditions, yields XVIII asdescribed by Pfeifer, M; Robinson, J. A. J Chem Soc. Chem. Commun. 1998,1977.

XVIII→XIX

xi: Cleavage of the tert.-butyl ester and tert-butyloxycarbonyl groups,conveniently using trifluoracetic acid in dichloromethane or 4Nhydrochloric acid in dioxane.

xii: The intermediate free amino acid formed is conveniently protectedwith reagents such as 9-fluorenylmethoxcarbonyl chloride or9-fluorenylmethoxcarbonyl succinimide using a base such as sodiumcarbonate or triethylamine in a suitable solvent or mixture of solventssuch as dioxane and water, or dichloromethane to yield XIX as describedby Pfeifer, M.; Robinson, J. A. J. Chem. Soc. Chem. Commun. 1998, 1977.

XX→XXI

i: Treatment of XX wit a dehydrating agent such as thionyl chloride in asuitable solvent such as methanol at an elevated temperature,conveniently at about 80° C.

ii: The intermediate is treated with a strong base such as lithiumdiisopropylamide or lithium hexamethyldisilylazide in a suitable solventsuch as tetrahydrofuran at low temperature, and with tert.-butylbromoacetate as described by Pfeifer, M.; Linden, A.; Robinson, J. A.Helv. Chim. Acta 1997, 80, 1513-1527.

iii: Saponification using a base such as lithium hydroxide in water anda suitable solvent such as methanol.

XXI→XXII

iv: Coupling of XXI with (2S,4R)-Z-hydroxy proline under standardpeptide coupling conditions, e.g. using reagents such as HBTU and HOBTand diisopropylethylamine as base in a suitable solvent such as DMF,yielding XXII as described by Pfeifer, M.; Linden, A.; Robinson, J. A.Helv. Chim. Acta 1997, 80, 1513-1527.

XXII→XXIII

v: Removal of the Z-group, e.g. by hydrogenation using H₂ and a catalystsuch as Palladium on charcoal in a suitable solvent such as ethylacetate.

XXIII→XXIV

vi: XXIII is converted into the corresponding tosylate according tostandard methods, e.g. by reaction with p-toluenesulfonyl chloride inpyridine.

vii: The intermediate tosylate is converted into the correspondingazide, e.g. by treatment with sodium azide in a suitable solvent such asDMF at an elevated temperature, conveniently at about 80° C.

viii: Reduction of the azide group to the amino group can convenientlybe performed with H₂ and a catalyst such as Palladium on charcoal in asuitable solvent such as ethyl acetate, or with triphenylphoshine.

ix: The intermediate free amino acid tert.-butylester is convenientlyprotected with reagents such as 9-fluorenylmethoxcarbonyl chloride or9-fluorenylmethoxcarbonyl succinimide using a base such as sodiumcarbonate or triethylamine in a suitable solvent or mixture of solventssuch as dioxane and water, or dichloromethane.

x: Acidolysis using e.g. trifluoracetic acid in dichloromethane givesconveniently XXIV as described by Pfeifer, M.; Linden, A.; Robinson, J.A. Helv. Chim. Acta 1997, 80, 1513-1527.

XXV→XXVI

i: Standard peptide coupling of VII with XXV under standard peptidecoupling conditions using reagent such as HBTU and HOBT and e.g.diisopropylethylamine as base in a suitable solvent such as DMF.

ii: Hydrogenation using H₂ and a catalyst such as Palladium on charcoalin solvents such as ethyl acetate, DMF and ethanol yields XXVI asdescribed by Beeli, R.; Steger, M.; Linden, A.; Robinson, J. A. Helv.Chim. Acta 1996, 79, 2235-2248.

XXVI→XXVII

iii: Oxidation of the OH group using reagents such as pyridine-sulfurtrioxide complex, Jones reagent or the Dess-Martin periodinane reagent.

iv: Wittig-Homer condensation of the intermediate ketone with(MeO)₂POCH₂COOMe and a base such as sodium hexamethyldisilylazide insolvents such as tetrahydrofuran or dimethoxyethane as described byBeeli, R.; Steger, M.; Linden, A.; Robinson, J. A. Helv. Chim. Acta1996, 79, 2235-2248.

v: Stereoselective hydrogenation of the double bond using e.g. H₂ and acatalyst such as Palladium on charcoal in a solvent such as ethanol, DMFand ethyl acetate.

vi: Hydrazinolysis of the intermediate phthalimide using e.g. hydrazinein a suitable solvent such as ethanol at an elevated temperature,conveniently at about 80° C.

vii: Saponification of the methyl ester group, e.g. by treatment with asuitable basic reagent such as lithium hydroxide in water and methanol.

viii: The intermediate free amino acid formed is conveniently protectedwith reagents such as 9-fluorenylmethoxcarbonyl chloride or9-fluorenylmethoxcarbonyl succinimide using a base such as sodiumcarbonate or triethylamine in a suitable solvent or mixture of solventssuch as dioxane and water, or dichloromethane to yield XXVII asdescribed by Beeli, R.; Steger, M.; Linden, A.; Robinson, J. A. Helv.Chim. Acta 1996, 79, 2235-2248.

XXVIII→XXIX

i: XXVIII can be synthesized according to P. Waldmeier, “Solid-supportedsynthesis of highly substituted xanthene-derived templates for thesynthesis of β-turn stabilized cyclic peptide libraries”, PhD-thesis,University of Zurich, 1996. For cleaving the phthalimide group XXVIII isconveniently submitted to hydrazinolysis, e.g. by treatment withhydrazine hydrate in a suitable solvent such as ethanol at an elevatedtemperature, e.g. at about 80° C.

ii: The intermediate aminonitrile is saponified, conveniently underbasic conditions, e.g. with aqueous sodium hydroxide in a suitablesolvent such as ethanol at an elevated temperature, conveniently underreflux, to yield XXIX.

XXIX→XXX

iii: The intermediate free amino acid formed is conveniently protectedwith reagents such as 9-fluorenylmethoxcarbonyl chloride or9-fluorenylmethoxcarbonyl succinimide using a base such as sodiumcarbonate or triethylamine in a suitable solvent or mixture of solventssuch as dioxane and water, or dichloromethane to yield XXX as describedby P. Waldmeier, “Solid-supported synthesis of highly substitutedxanthene-derived templates for the synthesis of β-turn stabilized cyclicpeptide libraries”, PhD-thesis, University of Zurich, 1996.

XXIX→XXXI

iv: Regioselective bromination of XXIX is performed preferably withbromine in acetic acid and dichloromethane. In a similar fashion R⁶═NO₂can be introduced by treatment with HNO₃ in acetic acid and R⁶═CH₂-NPtby treatment with hydroxymethyl phthalimide in H₂SO₄.

v: The amino group is conveniently Z-protected with reagents such asbenzyloxycarbonyl chloride or succinimide in a suitable solvent such asdioxane in presence of a base such as aqueous sodium hydroxide.

vi: The carboxylic acid group is esterified, preferably with DBU andmethyl iodide in DMF.

XXXI→XXXII

vii: Introduction of lower alkyl, substituted lower alkyl and arylsubstituents (R⁶), conveniently by Palladium(0)-catalyzedStille-(Stille, J. K. Angew. Chem.1986, 68, 504) and Suzuki-couplings(Oh-e, T.; Mijaura, N.; Suzuki, A. J. Org. Chem. 1993, 58, 2201).

viii: Removal of the Z-group, e.g. by hydrogenation using H₂ and acatalyst such as Palladium on charcoal in a suitable solvent such asethanol, DMF and ethyl acetate.

ix: Hydrolysis of the ester group, conveniently under acidic conditions,e.g. with 25% aqueous hydrochloric acid in a suitable solvent such asdioxane at an elevated temperature, preferably at about 100° C.

x: The intermediate free amino acid formed is conveniently protectedwith reagents such as 9-fluorenylmethoxcarbonyl chloride or9-fluorenylmethoxcarbonyl succinimide using a base such as sodiumcarbonate or triethylamine in a suitable solvent or mixture of solventssuch as dioxane and water, or dichloromethane to yield XXXII.

XXIX→XXXIII

i: Double ortho-bromination is performed preferably with excess brominein acetic acid and dichloromethane. In a similar fashion R⁶═R⁷═NO₂ canbe introduced by treatment with HNO₃ in acetic acid and R⁶═R⁷═CH₂-NPt bytreatment with hydroxymethyl phthalimide in H₂SO₄.

ii: The amino group is protected, conveniently Z-protected, withreagents such as benzyloxycarbonyl chloride or succinimide in a suitablesolvent such as dioxane in the presence of a base such as aqueous sodiumhydroxide.

iii: The carboxylic acid group is esterified, preferably with DBU andmethyl iodide in DMF to yield XXXIII.

XXXIII→XXXIV

iv: Introduction of lower alkyl, substituted lower alkyl and arylsubstituents (R⁶ ═R⁷), e.g. by Palladium(0)-catalyzed Stille-(Stille, J.K. Angew. Chem.1986, 68, 504) and Suzuki-couplings (Oh-e, T.; Mijaura,N.; Suzuki, A. J. Org. Chem. 1993, 58, 2201).

XXXIV→XXXV

v: Removal of the Z-group of XXIV, e.g. by hydrogenation using H₂ and acatalyst such as Palladium on charcoal in a suitable solvent such asethanol, DMF or ethyl acetate.

vi: Hydrolysis of the ester group, conveniently under acidic conditions,e.g. with 25% aqueous hydrochloric acid in a suitable solvent such asdioxane at an elevated temperature, conveniently at about 100° C.

vii: The intermediate free amino acid formed is conveniently protectedwith reagents such as 9-fluorenylmethoxcarbonyl chloride or9-fluorenylmethoxcarbonyl succinimide using a base such as sodiumcarbonate or triethylamine in a suitable solvent or mixture of solventssuch as dioxane and water, or dichloromethane to yield XXXV.

XXVIII→XXXVI

i: Cleavage of the methoxy groups of XXVII, preferably by treatment withan excess of boron tribromide in a suitable solvent such asdichloromethane.

ii: Hydrolysis of the cyano group under acidic conditions, preferablywith 25% aqueous hydrochloric acid in a suitable solvent such as dioxaneat an elevated temperature, conveniently at about 100° C.

iii: The resulting acid is treated with a dehydrating agent such asthionyl chloride in a suitable solvent such as dioxane yields XXXVI.

XXXVI→XXXVII

iv: Treatment of XXXVI with an appropriate triflating reagent,preferably trifluorosulfonic acid anhydride in the presence of a basesuch as 2,6-di-tert-butyl-pyridine in a suitable solvent such asdichloromethane.

v: Heating of the intermediate, conveniently in a suitable solvent suchas methanol.

XXXVII→XXXVIII

vi: Introduction of lower alkyl or aryl-lower alkyl (R⁴) by alkylation.

XXXVIII→XXXIX

vii: Introduction of lower alkyl or aryl (R⁵), conveniently byPalladium(0)-catalyzed Suzuki-coupling (Oh-e, T.; Mijaura, N.; Suzuki,A. J. Org. Chem. 1993, 58, 2201).

XXXIX→XL

viii: Hydrolysis of the ester group under acidic conditions,conveniently with 25% aqueous hydrochloric acid in a suitable solventsuch as dioxane at an elevated temperature, e.g. at about 100° C.

ix: Cleavage of the phthalimido group, conveniently by hydrazinolysis,e.g. with hydrazine hydrate in a suitable solvent such as ethanol.

x: The intermediate free amino acid formed is conveniently protectedwith reagents such as 9-fluorenylmethoxcarbonyl chloride or9-fluorenylmethoxcarbonyl succinimide using a base such as sodiumcarbonate or triethylamine in a suitable solvent or mixture of solventssuch as dioxane and water, or dichloromethane to yield XL.

XXXVI→XLI

i: Bromination of XXXVI using reagents such as bromine in a mixture ofacetic acid and dichloromethane at temperatures ranging from about 0° C.to about room temperature.

ii: Benzoylation of the hydroxy group using an appropriate acylatingagent such as benzoyl chloride or benzoic acid anhydride, a base such aspyridine or triethylamine and a suitable solvent such asdichloromethane.

XLI→XLII

iii: XLI is treated with methanol and a catalytic amount of an acidiccatalyst such as campher sulfonic acid under heating.

iv: Introduction of lower alkyl or aryl-lower alkyl (R⁴) by alkylationusing a base such as sodium hydride or potassium tert.-butoxide in asolvent such as tetrahydrofuran, dimethoxyethane or DMF.

XLII→XLIII

v: Lower alkyl, substituted lower alkyl and aryl substituents (R⁷) areintroduced, e.g. by Palladium(0)-catalyzed Stille-(Stille, J. K. Angew.Chem.1986, 68, 504) and Suzuki-couplings (Oh-e, T.; Mijaura, N.; Suzuki,A. J. Org. Chem. 1993, 58, 2201).

vi: For cleaving the benzyloxy group the intermediate is convenientlyheated with sodium cyanide adsorbed on aluminum oxide and methanol.

vii: Treatment with an appropriate triflating reagent, preferablytrifluorosulfonic acid anhydride, in the presence of a base such as2,6-di-tert-butyl-pyridine in a suitable solvent such asdichloromethane.

viii: Introduction of lower alkyl and aryl substituents (R⁵), e.g. byPalladium(0)-catalyzed Stille-(Stile, J. K. Angew. Chem.1986, 68, 504)and Suzuki-couplings (Oh-e, T.; Mijaura, N.; Suzuki, A. J. Org. Chem.1993, 58, 2201).

XLIII→XLIV

ix: Bromination under standard conditions such as using bromine inacetic acid and dichloromethane at temperatures ranging from about 0° C.to about room temperature.

XLIV→XLV

x: Lower alkyl substituted lower alkyl and aryl substituents (R⁶) areintroduced, e.g. by Palladium(0)-catalyzed Stifle-(Stille, J. K. Angew.Chem.1986, 68, 504) and Suzuki-couplings (Oh-e, T.; Mijaura, N.; Suzuki,A. J. Org. Chem. 1993, 58, 2201).

XLV→XLVI

xi: The ester group is hydrolyzed under acidic conditions, convenientlywith 25% aqueous hydrochloric acid in a suitable solvent such as dioxaneat an elevated temperature, e.g. at about 100° C.

xii: The phthalimido group is cleaved, e.g. by hydrazinolysis,conveniently with hydrazine hydrate in a suitable solvent such asethanol.

xiii: The intermediate free amino acid formed is conveniently protectedwith reagents such as 9-fluorenylmethoxcarbonyl chloride or9-fluorenylmethoxcarbonyl succinimide using a base such as sodiumcarbonate or triethylamine in a suitable solvent or mixture of solventssuch as dioxane and water, or dichloromethane to yield XLVI.

XLVII→XLVIII

i: 3,7-Dimethoxyphenothiazine is prepared and converted into XLVIIaccording to Müller, K; Obrecht, D.; Knierzinger, A.; Spiegler, C.;Bannwarth, W.; Trzeciak, A; Englert, G.; Labhardt, A.; Schönholzer, P.Perspectives in Medicinal Chemistry, Editor Testa, B.; Kyburz, E.;Fuhrer, W.; Giger, R., Weinheim, New York, Basel, Cambridge: VerlagHelvetica Chimica Acta, 1993, 513-531; Bannwarth, W.; Gerber, F.;Grieder, A.; Knierzinger, A; Müller, K.; Obrecht D.; Trzeciak, A. Can.Pat. Appl. CA2101599(131 pages). The benzyl group is cleaved off fromXLVII conveniently by hydrogenation, e.g. with H₂ and a catalyst such asPalladium on charcoal in a suitable solvent such as ethanol, DMF orethyl acetate.

ii: Introduction of lower alkyl (R) by alkylation using an appropriatealkylating agent (R⁹−X′; X′=OTf, Br, I) and strong bases such as sodiumamide in liquid nitrogen or sodium hydride in tetrahydrofuran, dioxan orDMF in the presence of a phase transfer catalyst such as TDA-I. In asimilar manner substituted lower alkyl (R⁹) can be introduced; thus, forexample R⁹═CH₂COOR¹⁰ and CH₂CH₂COOR¹⁰ can be introduced by treatmentwith the appropriate 2-halo acetic and, respectively, 3-halo propionicacid derivatives.

XLVIII→IL

iii: Cleavage of the methoxy groups of XLVIII, conveniently by treatmentwith an excess of boron tribromide in a suitable solvent such asdichloromethane at temperatures ranging from about −20° C. to about roomtemperature.

iv: For the introduction of lower alkyl, substituted lower alkyl oraryl-lower alkyl substituents (R⁸) the intermediate bis-phenolderivative is conveniently reacted with a reagent of the formulaR⁸−X′(X′=OTf, Br, I) in the presence of strong bases such as sodiumhydride in tetrahydrofuran, dioxan or DMF in the presence of a phasetransfer catalyst such as TDA-I.

XLVIII→L IL→LI

v: The cyano group of XLVIII and, respectively, IL is hydrolyzed,conveniently under acidic conditions, e.g. with 25% aqueous hydrochloricacid in a suitable solvent such as dioxane at an elevated temperature,e.g. at about 100° C.

vi: The phthalimide group of the intermediate is cleaved, convenientlyby hydrazinolysis, e.g. with hydrazine hydrate in a suitable solventsuch as ethanol.

vii: The free amino group is conveniently protected with reagents suchas 9-fluorenylmethoxcarbonyl chloride or 9-fluorenylmethoxcarbonylsuccinimide using a base such as sodium carbonate or triethylamine in asuitable solvent or mixture of solvents such as dioxane and water, ordichloromethane to yield L and, respectively, LI.

LIII→LIV

i: LII can be prepared from commercial resorufin and coverted into LIIIaccording to Müller, K.; Obrecht, D.; Knierzinger, A.; Spiegler, C.;Bannwarth, W.; Trzeciak, A.; Englert, G.; Labhardt, A.; Schönholzer, P.Perspectives in Medicinal Chemistry, Editor Testa, B.; Kyburz, E.;Fuhrer, W.; Giger, R., Weinheim, New York, Basel, Cambridge: VerlagHelvetica Chimica Acta, 1993, 513-531; Bannwarth, W.; Gerber, F.;Grieder, A.; Knierzinger, A.; Müller, K.; Obrecht. D.; Trzeciak, A. Can.Pat. Appl. CA2101599(131 pages). For splitting off the benzyl group LIIIis conveniently hydrogenated e.g. with H₂ and a catalyst such asPalladium on charcoal in a suitable solvent such as ethanol DMF or ethylacetate.

ii: Introduction of lower alkyl (R⁹) by alkylation with R⁹−X′(X′=OTf,Br, I) using strong bases such as sodium amide in liquid nitrogen orsodium hydride in tetrahydrofuran, dioxan or DMF in the presence of aphase transfer catalyst such as TDA-I to yield LIV. In a similar mannersubstituted lower alkyl (R⁹) can be introduced; thus, for example,R⁹═CH₂COOR¹⁰ and CH₂CH₂COOR¹⁰ can be introduced by treatment with theappropriate 2-halo acetic and, respectively, 3-halo propionic acidderivatives.

LIV→LV

iii: Cleavage of the methoxy groups of LIV, conveniently by treatmentwith excess boron tribromide in dichloromethane at temperatures rangingfrom about −20° to about room temperature.

iv: The intermediate bis-phenol derivative is preferably reacted withR⁸−X′(X′=OTf, Br, I) in the presence of strong bases such as sodiumhydride in tetrahydrofuran, dioxan or DMF in the presence of a phasetransfer catalyst such as TDA-I.

LIV→LVI LV→LVII

v: The cyano group of LIV and, respectively, LV is hydrolyzed underacidic conditions, e.g. with 25% aqueous hydrochloric acid in a suitablesolvent such as dioxane at an elevated temperature, conveniently atabout 100° C.

vi: The phthalimide group is cleaved, conveniently by hydrazinolysis,e.g. with hydrazine hydrate in suitable solvent such as ethanol.

vii: The free amino group is conveniently protected with reagents suchas 9-fluorenylmethoxcarbonyl chloride or 9-fluorenylmethoxcarbonylsuccinimide using a base such as sodium carbonate or triethylamine insuitable solvent or mixture of solvents such as dioxane and water, ordichloromethane to yield LVI and, respectively, LVII.

The following Examples illustrate the invention in more detail but arenot intended to limit its scope in any manner.

EXAMPLE 1 Preparation of a Single Compound of Formula I

1,4 g of 2-chlorotrityl chloride resin (1.25 mmol/g, 1.75 mmol) werefilled into a three necked flask. The resin was suspended in DCM (14 ml)and allowed to swell at room temperature under constant stirring. Theresin was treated with 1.25 g (1.077 equiv.) of Fmoc-Arg(Pmc)-OH and0.898 ml of diisopropylethylamine (DIPEA) in DCM (10 ml), the mixturewas shaken at 25° C. for 15 minutes, poured into the pre-swollen resinand stirred at 25° C. for 18 hours. The resin colour changed to purpleand the solution remained yellowish. The res in was washed extensivelyand dried at 40° C. under vacuum for 4 hours.

Yield: 2.379 gm Loading: 84%

The esterified resin was then subjected to the following synthesis cyclet 40 mg per reaction vessel.

Step Reagent Time 1 DCM, swell and wash 3 × min. 2 20% piperidine/DMF 1× 15 min. 3 DMF, wash and swell 5 × 1 mil. 4 4 equiv. Fmoc aminoacid/DMF + 1 × 120 min. 4 equiv. 1-benzotriazol-1-yl-tetramethylurounium hexafluoro phosphate (HBTU) + 4 equiv.1-hydroxybenzotriazole (HOBt) + 6 equiv. Diisopropylethylamine 5 DMF,wash 3 × 1 min. 6 isopropylalcohol, wash 2 × 1 min. 7 DCM, wash 2 × 1min.5 ml of the solvent were used in each step. Fmoc-Val-OH, Fmoc-Ile-OH,Fmoc-Glu(OtBu)-OH, Fmoc-L-Pro-OH, Fmoc-D-Pro-OH, Fmoc-Ile-OH,Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH and Fmoc-Lys-(Boc)-OH were coupledaccording to the above protocol.Cleavage of the Fully Protected Peptide Fragment

After completion of the synthesis, the peptide resin was suspended in 5ml of 1% TFA in DCM (v/v) and agitated for 10 minutes, whereupon theresin was filtered off and the filtrate was neutralized with pyridine (1equiv.). This procedure was repeated twice to ensure completion of thecleavage. The filtrate was evaporated to dryness and analyzed by reversephase (RP)-HPLC to monitor the efficiency of the linear peptidesynthesis.

Cyclization of theH-Lys(Boc)-Lys(Boc)-Pro-Ile-D-Pro-L-Pro-Glu(OtBu)-Ile-Val-Arg(Pmc)-OHlinear peptide [SEQ ID NO:31]

50 mg(0.0294 mmol) of the fully protected linear peptide were dissolvedin DMF (50 ml, conc. 1 mg/ml). Then 33.5 mg (0.0882 mMol), 3 equiv.) ofHATU, 12.0 mg (0.0882 mMol), 3 eq) of HOAt and 5 ml of DIPEA (1% v/v)were added and the mixture was stirred at 20° C. for 16 hours andsubsequently concentrated in a vacuum. The residue was partitionedbetween dichloromethane (DCM) and H₂O/CH₃CN (90:10). The DCM phase wasevaporated to yield the pure filly protected cyclic peptide.

Deprotection of the Cyclic Peptide:

The amorphous powder obtained was dissolved in 2 ml of the cleavagemixture containing 95% trifluoroacetic acid, 2.5% water and 2.5%triisopropyl siliane (TIS). The mixture was left to stand at 20° C. for2 hours and then concentrated in a vacuum. The residue was trituratedwith diethyl ether, and 20 mg of compound I [SEQ ID NO:32] were obtainedas a white colored powder.

C₅₅H₉₅ N₁₅ O₁₂, MW 1158.5 MS(ESI): 580.02 (M + 2H⁺)²⁺, 387.02 (M +3H⁺)³⁺ HPLC-RT(min.): 7.51Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 5%acetonitril/water(0.1% trifluoroacetic acid to 100% acetonitril in 15minutes; stay constant for 5 minutes and return to 5%acetonitril/water(0.1% trifluoroacetic acid) in 5 minutes.

EXAMPLE 2 Preparation of a Single Compound of Formula I

By a procedure analogous to that described in Example 1, Fmoc-Val-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH,Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH and Fmoc-Lys(Boc)-OHwere coupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 2 [SEQ ID NO:33]:

C₆₂H₉₅ N₁₅ O₁₄, MW 1274.5 MS(ESI): 638.4 (M + 2H⁺)²⁺, 424.8.02 (M +3H⁺)³⁺ HPLC-RT(min.): 8.59Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 5%acetonitril/water(0.1% trifluoroacetic acid to 100% acetonitril in 15minutes; stay constant for 5 minutes and return to 5%acetonitril/water(0.1% trifluoroacetic acid) in 5 minutes.

EXAMPLE 3 Preparation of a Single Compound of Formula I

By a procedure analogous to that described in Example 1, Fmoc-Val-OH,Fmoc-Trp-OH, Fmoc-Glu(OtBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH,Fmoc-Ile-OH, Fmoc-Trp-OH, Fmoc-Lys(Boc)-OH and Fmoc-Lys(Boc)-OH werecoupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 3 [SEQ ID NO:34]:

C₆₆H₉₇ N₁₇ O₁₂, MW 1320.6 MS(ESI): 1321.6 (M + H⁺)⁺ HPLC-RT(min.): 9.04Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No 8111346, Batch 8023); gradient 5%acetonitril/water(0.1% trifluoroacetic acid to 100% acetonitril in 15minutes; stay constant for 5 minutes and return to 5%acetonitril/water(0.1% trifluoroacetic acid) in 5 minutes.

EXAMPLE 4 Preparation of a Single Compound of Formula I

By a procedure analogous to that described in Example 1, Fmoc-Val-OH,Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH,Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH and Fmoc-Lys(Boc)-OH werecoupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 4 [SEQ ID NO:35]:

C₅₀H₈₇ N₁₅ O₁₂, MW 1090.5 MS(ESI): 546.15.4 (M + 2H⁺)²⁺, 364.3 (M +3H⁺)³⁺ HPLC-RT(min.): 12.51Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 5%acetonitril/water(0.1% trifluoroacetic acid to 100% acetonitril in 15minutes; stay constant for 5 minutes and return to 5%acetonitril/water(0.1% trifluoroacetic acid) in 5 minutes.

EXAMPLE 5 Preparation of a Single Compound of Formula I

By a procedure analogous to that described in Example 1, Fmoc-Val-OH,Fmoc-Ser(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH,Fmoc-Ile-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Lys(Boc)-OH and Fmoc-Lys(Boc)-OHwere coupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 5 [SEQ ID NO:36]:

C₅₀H₈₇ N₁₅ O₁₄, MW 1122.3 MS(ESI): 562.15 (M + 2H⁺)²⁺, 375.3 (M + 3H⁺)³⁺HPLC-RT(min.): 5.74Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 5%acetonitril/water(0.1% trifluoroacetic acid to 100% acetonitril in 15minutes; stay constant for 5 minutes and return to 5%acetonitril/water(0.1% trifluoroacetic acid) in 5 minutes.

EXAMPLE 6 Synthesis of a Library of Compounds of Formula I for Mimickingthe PDGF-loop-III on a Diproline Template and Testing thereof in aSolid-Phase Assay

1.Target peptides x₁-x₄: variable amino acid residues (x) ValArgLysLys(VRKK) [SEQ ID NO:1]: constant amino acid residues ^(D)Pro^(L)Pro:template

x₁ ¹⁻⁴: Glu, Tyr, Trp, Ala x₂ ¹⁻⁶: Ile, Tyr, Trp, Ala, Ser, Lys x₃ ¹⁻⁶:Pro, Tyr, Trp, Ala, Ser, Lys x₄ ¹⁻⁴: Ile, Tyr, Trp, Ala

TABLE 1 The sequence of the 24 target cyclic peptides (the first fivecorrespond- ing to those obtained according to Examples 1-5) x₁ ¹x₄ ¹ x₁²x₄ ² x₁ ³x₄ ³ x₁ ⁴x₄ ⁴ (E-I) (Y-Y) (W-W) (A-A) x₂ ¹x₃ ¹ 1 7 13 19 (I-P)^(L)P-E-I-V-R ^(L)P-Y-I-V-R ^(L)P-W-I-V-R ^(L)P-A-I-V-R ^(D)P-I-P-K-K^(D)P-Y-P-K-K ^(D)P-W-P-K-K ^(D)P-A-P-K-K [SEQ ID [SEQ ID [SEQ ID [SEQID NO:32] NO:39] NO:45] NO:51] x₂ ²x₃ ² 2 8 14 20 (Y-Y) ^(L)P-E-Y-V-R^(L)P-Y-Y-V-R ^(L)P-W-Y-V-R ^(L)P-A-Y-V-R ^(D)P-I-Y-K-K ^(D)P-Y-Y-K-K^(D)P-W-Y-K-K ^(D)P-A-Y-K-K [SEQ ID [SEQ ID [SEQ ID [SEQ ID NO:33]NO:40] NO:46] NO:52] x₂ ³x₃ ³ 3 9 15 21 (W-W) ^(L)P-E-W-V-R^(L)P-Y-W-V-R ^(L)P-W-W-V-R ^(L)P-A-W-V-R ^(D)P-I-W-K-K ^(D)P-Y-W-K-K^(D)P-W-W-K-K ^(D)P-A-W-K-K [SEQ ID [SEQ ID [SEQ ID [SEQ ID NO:34]NO:41] NO:47] NO:53] x₂ ⁴x₃ ⁴ 4 10 16 22 (A-A) ^(L)P-E-A-V-R^(L)P-Y-A-V-R ^(L)P-W-A-V-R ^(L)P-A-A-V-R ^(D)P-I-A-K-K ^(D)P-Y-A-K-K^(D)P-W-A-K-K ^(D)P-A-A-K-K [SEQ ID [SEQ ID [SEQ ID [SEQ ID NO:35]NO:42] NO:48] NO:54] x₂ ⁵x₃ ⁵ 5 11 17 23 (S-S) ^(L)P-E-S-V-R^(L)P-Y-S-V-R ^(L)P-W-S-V-R ^(L)P-A-S-V-R ^(D)P-I-S-K-K ^(D)P-Y-S-K-K^(D)P-W-S-K-K ^(D)P-A-S-K-K [SEQ ID [SEQ ID [SEQ ID [SEQ ID NO:36]NO:43] NO:49] NO:55] x₂ ⁶x₃ ⁶ 6 12 18 24 (K-K) ^(L)P-E-K-V-R^(L)P-Y-K-V-R ^(L)P-W-K-V-R ^(L)P-A-K-V-R ^(D)P-I-K-K-K ^(D)P-Y-K-K-K^(D)P-W-K-K-K ^(D)P-A-K-K-K [SEQ ID [SEQ ID [SEQ ID [SEQ ID NO:38]NO:44] NO:50] NO:56]2. Experimental Procedures:2.1. Synthesis of Protected Linear Peptides

The first amino acid Fmoc-Arg(Pmc)-OH (1 eq.) was linked to2-chlorotrityl chloride resin (Polyphor, 1.25 mmol/g) with 3 eq. DIEA inDCM overnight, the attachment was ca.85%. The linear peptides wereassembled using standard Fmoc chemistry, 4 eq. each of amino acids, ofHBTU and HOBt and 6 eq. of DIEA in DMF being used and the coupling timebeing 1.5-2 h The protected linear peptides were cleaved from the resinwith 1% TFA in DCM (2×10 min.) and neutralized with pyridine (1 eq.),then the solvent was evaporated.

2.2. Cyclisation of Protected Linear Peptides

The protected linear peptide (without purification) was directlycyclized at a concentration of 1.0 mg/ml in DMF using HATU (3 eq.), HOAt(3 eq.) and DIEA (1% v/v) for 16 h. Then DMF and DIEA were evaporated,the residue was dissolved in DCM, the solution was extracted withH₂O/CH₃CN(90:10), and afterwards the DCM was removed.

2.3. Deprotection of the Cyclized Peptides

The cyclization product was treated with 95% TFA, 2.5% H₂O and 2.5% TISfor 2 h, then most of the TFA was evaporated. Et₂O was added toprecipitate the product. After centrifugation, the ether was carefullyremoved and the final product was obtained after drying under reducedpressure. Depending on its purity, the product was purified bypreparative HPLC.

2.4. Solid-phase Assay

Direct immobilization of platelet-derived growth factor β-receptor(PDGFR-β) was performed by overnight incubation in immunosorbent 96-wellplates (Nunc) at 4° C. using 100 ng of purified protein in 100 μl of 15mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6. Plates were washed once withTris-buffered saline (TBS, 20 mM Tris-HCL, 150 mM Na Cl, pH 7.4), andnonspecific adsorption was blocked by at least 1 h of incubation withTBS plus 1% bovine serum albumin (BSA). Following washing with TBS plus0.1% Tween, 3000 cpm of ¹²⁵I-PDGF-BB and increasing amounts of unlabeledPDGF-BB or the peptides to be tested were added to duplicate wells andincubated for 3 h at room temperature in TBS plus 0.1% Tween, 1 mMCaCl₂, 1 mM MgCl₂, and 1% BSA. The plates were washed three times withTBS plus 0.1% Tween, and the bound ligand was removed with 0.1 M citricacid, pH 2.5, before counting in a γ-counter.

3. Results

The cyclic peptides were analyzed and purified by preparative HPLC(dual-pump Pharmacia system with Waters RCM-μBondapak®-C₁₈-cartridges,10 μm 300 A 25×100 mm for prep. and 8×100 mm for anal., with flow ratesof 8 and 2 ml/mim, respectively; UV detection at 226 and 278 nm), thenMS, NMR(600 MHz,1 H) and CD. Solid-phase assays were run, as describedin 2.4.

4. Discussion

4.1. Linear peptides were analyzed by HPLC, all of the 24 compoundsturned out to be pure, >95% indicating that the assembling of aminoacids worked performed reliably.

4.2 Cyclized peptides

-   a) The linear peptides cleaved from resin, neutralized with pyridine    to form pyridine salts, which needed not to be purified before their    cyclization.-   b) Different concentrations of peptides for cyclization were    compared, 1 mg, 2 mg, 5 mg, 10 mg, 20 mg/ml DMF, the 1 mg/ml    concentration gave the best result-   c) The purities of the crude products are shown in Table 2.

4.3. Solid-phase assay

The IC₅₀-values are shown in Table 2. The differences in IC₅₀-valuesbetween the crude and purified peptides were only marginal.

TABLE 2 Summary of Examples 1-24 Tar- ESI-MS Retention get [M+H⁺]⁺; timeof Purity of pep- Formula [M+2H⁺]²⁺; HPLC crude Assay tide M.W.[M+3H⁺]³⁺; (min) product I₅₀ (μM)  1 C₅₅H₉₅N₁₅O₁₂ 580.02; 387.02 7.5195% 2200 1158.5  2 C₆₂H₉₅N₁₅O₁₄ 1274.8; 8.59 95% 2000 1274.5 638.01;425.75  3 C₆₆H₉₇N₁₇O₁₂ 1320.81; 9.04 80% 1500 1320.6 661.06; 441.11  4C₅₀H₈₇N₁₅O₁₂ 1090.54; 90% 12.5 <2500 1090.3 545.83; 364.26  5C₅₀H₈₇N₁₅O₁₄ 1122.71; 5.74 95% 2500 1122.3 562.07; 375.05  6C₅₆H₁₀₁N₁₇O₁₂ 1205.7; 9.90 95% <2500 1204.5 603.18; 402.58  7C₆₂H₉₅N₁₅O₁₂ 621.90; 414.89 9.14 95% 1500 1242.5  8 C₆₉H₉₅N₁₅O₁₄ 679.82;453.61 9.42 95% 800 1358.6  9 C₇₃H₉₇N₁₇O₁₂ 1404.83; 9.71 65% 500 1404.7703.08; 469.16 10 C₅₇H₈₇N₁₅O₁₂ 1174.73; 9.09 90% 2000 1174.4 587.97;392.39 11 C₅₇H₈₇N₁₅O₁₄ 1206.75; 9.10 90% 2000 1206.4 604.02; 403.01 12C₆₃H₁₀₁N₁₇O₁₂ 1288.92; 8.59 90% 1500 1288.6 645.06; 430.47 13C₆₆H₉₇N₁₇O₁₀ 1288.82; 8.27 95% 260 1288.6 645.08; 430.47 14 C₇₃H₉₇N₁₇O₁₂1405.0; 9.26 90% 170 1404.7 703.09; 469.08 15 C₇₇H₉₉N₁₉O₁₀ 1451.06;10.35  20% 1450.8 726.06; 484.42 16 C₆₁H₈₉N₁₇O₁₀ 1220.87; 9.81 90% 8001220.5 611.03; 407.74 17 C₆₁H₈₉N₁₇O₁₀ 1252.85; 9.84 90% 800 1252.5627.03; 418.46 18 C₆₇H₁₀₃N₁₉O₁₀ 1334.78; 9.10 90% 500 1334.7 668.15;445.80 19 C₅₀H₈₇N₁₅O₁₀ 1058.84; 7.86 95% <2500 1058.3 530.03; 353.69 20C₅₇H₈₇N₁₇O₁₀ 1174.71; 8.85 30% 2000 1174.4 588.11; 407.78 21C₆₁H₈₉N₁₇O₁₀ 1220.91; 8.85 30% 2000 1220.5 611.16; 407.78 22C₄₅H₇₉N₁₅O₁₂ 495.7 6.77 85% <2500  990.2 23 C₄₅H₇₉N₁₅O₁₂ 511.94 7.12 85%2500 1022.2 24 C₅₁H₉₃N₁₇O₁₀ 553.12 6.86 90% 2500 1104.4Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5 Protect 1(Ser. No. 8111346, Batch 8023); gradient: 5% acetonitril/water(0.1%trifluoroacetic acid to 100% acetonitril in 15 ) minutes; stay constantfor 5 minutes and return to 5% acetonitril/water(0.1% trifluoroaceticacid) in 5 minutes.

EXAMPLES 25-40

The following Examples describe the application of the process to thesynthesis of 6-mer, 8-mer, 10-mer, 12-mer, 14-mer and 16-mer β-hairpinloop mimetics incorporating three different templates and a common keymotif -k¹-x¹-template-x²-k²-[SEQ ID NO:57] where x¹=Y, F, K or W, x²=Y,k¹=K and k²=E. Due to the β-hairpin structure x¹ and x² are lying on thesame side of the β-sheet and form a hydrophobic patch. Such motifs arepresent e.g. in various chemokines (see Tarby, C. M.; Saunders, J. DrugDiscovery Today 1999, 4, 80-92; Ponath, P. D. Exp. Opin. Invest. Drugs1998, 7, 1-16).

1. Synthesis of(2S,6S,8aS)-8a-{[(tert-butyl)oxycarbonyl]methyl}perhydro-5,8-dioxo-{[(9H-fluoren-9-yl)methoxycarbonyl]amino}-pyrrolo[1,2-a]pyrazine-6-aceticacid (template b1):

To a stirred solution of 250 mg (0.414 mmol) of allyl{(2S,6S,8aS)-8a-[(tert-butyl)oxycarbonyl]methyl}perhydro-5,8-dioxo-{[(9H-fluoren-9-yl)methoxycarbonyl]amino}-pyrrolo[1,2-a]pyrazin-6-acetatein a degassed mixture of dichloromethane/methanol (9:1, 3 ml) were addedunder argon 25 mg (0.0216 mmol) oftetrakis(triphenylphosphine)palladium, 0.05 ml of acetic acid and 0.025ml of N-methylmorpholin. The reaction mixture was stirred for 48 hoursat room temperature and poured onto water and dichloromethane. Theorganic phase was dried (MgSO4), evaporated and the residuechromatographed on SiO₂ with dichloromethane/methanol (9:1) to yield 180mg (77/o) of(2S,6S,8aS)-8a-{[(tert-butyl)oxycarbonyl]methyl}perhydro-5,8-dioxo-{[(9H-fluoren-9-yl)methoxycarbonyl]amino}-pyrrolo[1,2-a]pyrazine-6-aceticacid (template b1) as a white powder.

¹H-NMR(300 MHz, DMSO-d₆): 8.30 (s, 1H); 7.88 (d, J=7.2, 2H); 7.67 (d,J=7.4, 2H); 7.62 br.s, 1H); 7.41 (t, 7.2, 2H); 7.33 (t, J=7.4, 2H);4.35-4.2 (m, 5H); 355 (br.d, 3=63, 2H); 2.8-2.55 (m, 3H); 2.45-2.25 (m,2H); 2.1-1.95 (m, 1H); 1.35 (s, 9H); MS(ESI): 586.1 (M+Na)⁺, 564.1(M+H)⁺.

2. Synthesis of Linear Peptides:

The first amino acid Fmoc-Arg(Pmc)-OH (1 eq.) was linked to2-chlorotrityl chloride resin (Polyphor, 1.25 mmol/g) with 3 eq. DIEA inDCM overnight, the attachment was ca.80%. The linear peptides wereassembled using standard Fmoc chemistry, 4 eq. each of amino acids andof the template (or, if appropriate, of Fmoc-^(L)Pro-OH and ofFmoc-^(D)Pro-OH), 4 eq. each of HBTU and HOBt and 6 eq. of DIEA in DMFbeing used and the coupling time being 1.5-2 h. The protected linearpeptides were cleaved from the resin with 1% TFA in DCM (4×10 min.) andneutralized with pyridine (1 eq.), then the solvent was evaporated.

3. Cyclisation of the Linear Peptides

The protected linear peptide (without purification) was directlycyclized at a concentration of 1.0 mg/ml in DMF using HATU (3 eq.), HOAt(3 eq.) and DIEA (1% v/v) for 16 h. Then DMF and DIEA were evaporated,the residue was dissolved in DCM, the solution was extracted withH₂O/CH₃CN (90:10), and afterwards the DCM was removed.

4. Deprotection of the Cyclized Peptides

The cyclization product was treated with 95% TFA, 2.5% H₂O and 2.5% TISfor 2 h, then most of the TFA was evaporated. Et₂O was added toprecipitate the product. After centrifugation, the ether was carefullyremoved and the final product was obtained after drying under reducedpressure. Depending on its purity, the product was purified bypreparative HPLC. The following templates were used.

EXAMPLE 25

By a procedure analogous to that described in Example I, Fmoc-Ala-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH and Fmoc-Arg(Pmc)-OHwere coupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 25 [SEQ ID NO:58].

MW: C₅₇H₈₅N₁₇O₁₄, [1232.30] MS (ESI): 616.72 [M + 2H⁺]²⁺ HPLC-RT (min.):10.83Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 26

By a procedure analogous to that described in Example 1, Fmoc-Ala-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH,Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH and Fmoc-Arg(Pmc)-OH werecoupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 26 [SEQ ID NO:59].

MW: C₅₇H₈₅N₁₇O₁₃, [1216.41] MS (ESI): 608.8 [M + 2H⁺]²⁺ HPLC-RT (min.):8.27Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 27

By a procedure analogous to that described in Example 1, Fmoc-Ala-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH,Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH and Fmoc-Arg(Pmc)-OHwere coupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 27 [SEQ ID NO:60].

MW: C₅₄H₈₈N₁₈O₁₃, [1197.4] MS (ESI): 599.4 [M + 2H⁺]²⁺ HPLC-RT (min.):8.85Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 28

By a procedure analogous to that described in Example 1, Fmoc-Ala-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH,Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH and Fmoc-Arg(Pmc)-OHwere coupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 28 [SEQ ID NO:61].

MW: C₅₉H₈₇N₁₈O₁₃, [1256.4] MS (ESI): 628.50 [M + 2H⁺]²⁺, 419.20 [M +3H⁺]³⁺ HPLC-RT (min.): 9.16Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 29

By a procedure analogous to that described in Example 1,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Tyr(OtBu)-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH andFmoc-Arg(Pmc)-OH were coupled to the Fmoc-Arg(Pmc)-OH loaded resin,cleaved, cyclised and deprotected to yield compound 29 [SEQ D NO:62].

MW: C₈₆H₁₂₂N₂₂O₂₂, [1816] MS (ESI): 908 [M + 2H⁺]²⁺, 606.2 [M + 3H⁺]³⁺HPLC-RT (min.): 8.40Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 1005Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 30

By a procedure analogous to that described in Example 1,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu-(OtBu)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH andFmoc-Arg(Pmc)-OH were coupled to the Fmoc-Arg(Pmc)-OH loaded resin,cleaved, cyclised and deprotected to yield compound 30 [SEQ ID NO:63].

MW: C₈₃H₁₂₅N₂₃O₂₁, [1781] MS (ESI): 594.6 [M + 3H⁺]³⁺ HPLC-RT (min.):9.04Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 31

By a procedure analogous to that descried in Example 1,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH andFmoc-Arg(Pmc)-OH were coupled to the Fmoc-Arg(Pmc)-OH loaded resin,cleaved, cyclised and deprotected to yield compound 31 [SEQ ID NO:64]

MW: C₈₃H₁₂₅N₂₃O₂₁, [1721] MS (ESI): 891.15 [M + 2H⁺]²⁺, 594.85 [M +3H⁺]³⁺ HPLC-RT (min.): 9.84Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 32

By a procedure analogous to that described in Example 1, Fmoc-Ala-OH,Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Ala-OH,Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, and Fmoc-Arg(Pmc)-OH werecoupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 32 [SEQ ID NO:65]

MW: C₁₁₀H₁₅₀N₂₆O₂₆, [2252.4] MS (ESI): 751.93 [M + 3H⁺]³⁺ HPLC-RT(min.): 9.42Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 33

By a procedure analogous to that described in Example 1, Fmoc-Ala-OH,Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-^(L)Pro-OH, Fmoc-^(D)Pro-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, and Fmoc-Arg(Pmc)-OHwere coupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 33 [SEQ ID NO:66].

MW: C₁₀₄H₁₅₆N₂₈O₂₆, [2214.5] MS (ESI): 738.10 [M + 3H⁺]³⁺ HPLC-RT(min.): 13.46Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 34

By a procedure analogous to that described in Example 1, Fmoc-Ala-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Template f1, Fmoc-Tyr(tBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, and Fmoc-Arg(Pmc)-OH were coupled to theFmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised and deprotected toyield compound 34 [SEQ ID NO:67].

MW: C₆₇H₉₁N₁₆O₁₆, [1376.5] MS (ESI): 689.02 [M + 2H⁺]²⁺ HPLC-RT (min.):9.87Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10% acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 35

By a procedure analogous to that described in Example 1, Fmoc-Ala-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Template f1, Fmoc-Phe-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, and Fmoc-Arg(Pmc)-OH were coupled to theFmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised and deprotected toyield compound 35 [SEQ ID NO:68].

MW: C₆₇H₉₁N₁₆O₁₅, [1360.14] MS (ESI): 681.44 [M + 2H⁺]²⁺, 454.77 [M +3H⁺]³⁺ HPLC-RT (min.): 9.68Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 36

By a procedure analogous to that described in Example 1,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Tyr(tBu)-OH, Template f1, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Arg(Pmc)-OHwere coupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 36 [SEQ ID NO:69].

MW: C₉₃H₁₃₁N₂₂O₂₃, [1925.19] MS (ESI): 643.28 [M + 3H⁺]³⁺ HPLC-RT(min.): 8.85 min.Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 37

By a procedure analogous to that described in Example 1,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Template b1, Fmoc-Trp(Boc)-OH,Fmoc-Lys(Boc)-OH and Fmoc-Arg(Pmc)-OH were coupled to theFmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised and deprotected toyield compound 37 [SEQ ID NO:70].

MW: C₅₄H₇₄N₁₇O₁₄, [1185.18] MS (ESI): 593.83 [M + 2H⁺]²⁺ HPLC-RT (min.):11.23Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 38

By a procedure analogous to that described in Example 1,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Template f1, Fmoc-Phe-OH,Fmoc-Lys(Boc)-OH, Fmoc-Tyr(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH andFmoc-Arg(Pmc)-OH were coupled to the Fmoc-Arg(Pmc)-OH loaded resin,cleaved, cyclised and deprotected to yield compound 38 [SEQ ID NO:71].

MW: C₉₆H₁₂₉N₂₁O₂₂, [1929.23] MS (ESI): 644 [M + 3H⁺]³⁺, 483.11 [M +4H⁺]⁴⁺ HPLC-RT (min.): 9.22

Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 1005Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 39

By a procedure analogous to that describe in Example 1,Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Template b1, Fmoc-Tyr(tBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Tyr(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Glu(OtBu)-OH and Fmoc-Arg(Pmc)-OH were coupled to theFmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised and deprotected toyield compound 39 [SEQ ID NO:72].

MW: C₁₀₅H₁₃₈N₂₅O₂₉, [2214.3] MS (ESI): 737.76 [M + 3H⁺]³⁺ HPLC-RT(min.): 13.26Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient: 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

EXAMPLE 40

By a procedure analogous to that described in Example 1,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Tyr(tBu)-OH, Template f1, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Arg(Pmc)-OHwere coupled to the Fmoc-Arg(Pmc)-OH loaded resin, cleaved, cyclised anddeprotected to yield compound 40 [SEQ ID NO:73].

MW: C₉₃H₁₃₁N₂₂O₂₃, [1926.22] MS (ESI): 643.01 [M + 3H⁺]³⁺, 482.35 [M +4H⁺]⁴⁺ HPLC-RT (min.): 8.99Conditions: Analytical HPLC-conditions: column CC 250/4 Nucleosil 100-5Protect 1 (Ser. No. 8111346, Batch 8023); gradient 10% acetonitril/90%water(containing 0.1% trifluoroacetic acid) to 100% acetonitril in 15minutes; stay constant for 4 minutes and return to 10%acetonitril/water(0.1% trifluoroacetic acid) in 4 minutes.

1. A process for the manufacture of compounds of the general formula

wherein Z is a chain of n α-amino acid residues which, if their α-C atomis asymmetric, have L-configuration, n being an integer from 4 to 20,the positions of said amino acid residues in said chain being countedstarting from the N-terminal amino acid;

is one of the groups of formulae

and of salts thereof, which process is capable of being carried out asparallel array synthesis to yield libraries of numerous compounds offormula I in high yields and defined purities and which comprises yieldlibraries of numerous compounds of formula I in high yields and definedpurities and which comprises (a) coupling a solid support derived frompolystyrene crosslinked with divinylbenzene which is functionalized bymeans of a 2-chlorotrityl linker with an appropriately N-protectedderivative of that amino acid which in the desired end-product is inposition n/2, n/2+1 or n/2−1 if n is an even number and, respectively,in position n/2+½ or n/2−½ if n is an odd number, any functional groupwhich may be present in said N-protected amino acid derivative beinglikewise appropriately protected; (b) removing the N-protecting groupfrom the product thus obtained; (c) coupling the product thus obtainedwith an appropriately N-protected derivative of that amino acid which inthe desired end-product is one position nearer the N-terminal amino acidresidue, any functional group which may be present in said N-protectedamino acid derivative being likewise appropriately protected; (d)removing the N-protecting group from the product thus obtained; (e)repeating, if necessary, steps (c) and (d) until the N-terminal aminoacid residue has been introduced; (f) coupling the product thus obtainedwith a compound of the general formula

wherein

is as defined above and X is an N-protecting group or, if

is to be group (a), above, alternatively (fa) coupling the productobtained in step (d) or (e) with a compound of the general formula III

wherein R¹ and X are as defined above; (fb) removing the N-protectinggroup from the product thus obtained; and (fc) coupling the product thusobtained with an appropriately N-protected derivative of D-proline; (g)removing the N-protecting group from the product obtained in step (f) or(fc); (h) coupling the product thus obtained with an appropriatelyN-protected derivative of that amino acid which in the desiredend-product is in position n, any functional group which may be presentin said N-protected amino acid derivative being likewise appropriatelyprotected; (i) removing the N-protecting group from the product thusobtained; (j) coupling the product thus obtained with an appropriatelyN-protected derivative of that amino acid which in the desiredend-product is one position farther away from position n, any functionalgroup which may be present in said N-protected amino acid derivativebeing likewise appropriately protected; (k) removing the N-protectinggroup form the product thus obtained; (l) repeating, if necessary, steps(j) and (k) until all amino acid residues have been introduced; (m)detaching the product thus obtained from the solid support; (n)cyclising the product cleaved from the solid support by means ofO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (“HATU”)/7-aza-1-hydroxybenzotriazole (“HOAt”); and(o) removing any protecting groups present on functional groups of anymembers of the chain of amino acid residues and, if desired, anyprotecting group(s) which may in addition be present in the molecule. 2.A process according to claim 1 wherein X and the N-protecting group ofthe amino acid derivatives is 9-fluorenylmethoxycarbonyl (Fmoc).
 3. Amodification of the process according to claim 1 for the manufacture ofenantiomers of the compounds of formula I as defined in claim 1 in whichall amino acids which have an asymmetric α-carbon atom are used in theirD-Form and the enantiomer of a template corresponding to structure (a),(b), (c), (d) or (e) or a template corresponding to formula (f), (g) or(h) is used in step (f) and, respectively, the enantiomer of a compoundof formula III is used in step (fa) and a derivative of L-proline isused in step (fc).
 4. A process according to claim 1, further comprisingconverting the product thus obtained into a salt or converting a saltthus obtained into the corresponding free compound of formula I or intoa different salt.